Abstract Glaucoma is a heterogenous group of optic neuropathies and a leading cause of irreversible vision loss and blindness worldwide. Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma and is often associated with elevated intraocular pressure (IOP), a leading causative risk factor for glaucoma. Elevated IOP is due to damage to the trabecular meshwork (TM) that increases resistance to aqueous humor outflow. Current glaucoma treatments do not address the underlying primary pathophysiological mechanisms and the disorder can continue to progress despite treatment. To truly cure glaucoma, there is critical need to permanently inhibit or reverse the glaucomatous damage to the TM and restore normal TM cell functions. New technologies, specifically CRISPR/Cas9 genome editing, allow investigators to directly modify the genes associated with pathogenic damage. We recently demonstrated the feasibility of targeting the mutant myocilin gene using CRISPR-Cas9 genome editing to prevent and reverse glaucoma in myocilin-associated POAG in mice and in human donor eyes. It is our goal to develop genome editing methods targeting the TM for the treatment of myocilin-associated POAG, and importantly the treatment of POAG in general. We have a unique opportunity to cure glaucoma by inhibiting the ongoing disease pathology by targeting common pathological pathways associated with general POAG. We have previously linked the pathological role of endoplasmic reticulum (ER) stress to the glaucomatous TM damage and IOP elevation in mouse models of ocular hypertension and in human donor eyes. Specifically, we have shown that ER-stress induced ATF4 and CHOP are associated with glaucomatous TM damage and independent inhibition of ATF4 and CHOP can be a novel treatment strategy for general POAG. Therefore, we propose to target this common underlying mechanism using the CRISPR-Cas9 system. It is our overall goal to design AAV vectors encoding CRISPR/Cas9 targeting MYOC, CHOP and ATF4 to selectively knockout these genes in the TM and lower IOP in mouse ocular hypertension models, as well as in ex vivo human perfusion organ cultured anterior segments. We hypothesize that genome editing knockout of MYOC, CHOP and ATF4 using AAV.CRISPR/Cas9 vectors targeting the TM will prevent and reverse elevated IOP in glaucoma. We will use AAV vectors because these agents have been used for gene therapy in patients with specific eye diseases. In this proposal, we will first determine the most efficient AAV vector(s) for TM tropism and design efficient CRISPR/Cas9 constructs that effectively knockdown MYOC, CHOP and ATF4 in the TM. We will also evaluate CRISPR-Cas9 off-target effects in human donor eyes. Second, we determine the efficacy of AAV.cr. MYOC, AAV.cr. CHOP and AAV.cr. ATF4 knockouts in the TM and the ability of these knockouts to reduce elevated IOP in vivo in mice and ex vivo in human perfusion organ cultured anterior segments. Our approach will revolutionize glaucoma therapy by directly interfering with glaucomatous damage to the TM and provide a one-time therapy to effectively treat glaucoma resulting from elevated IOP.